The general aim of this project is to identify several cellular factors that contribute to the assembly and cell surface expression of the nicotinic acetylcholine receptor expressed by transfection in mammalian cells. The first aim is to determine whether the adherence of the chaperone protein calnexin to unassembled receptor subunits acts to retain receptor subunits in the endoplasmic reticulum until assembly occurs. Using a truncated calnexin cDNA that lacks its ER retention signal and is transported to the cell surface, truncated calnexin cDNA and receptor subunit cDNA will be co-transfected and the cellular localization of receptor subunits will be characterized. The second aim is to determine whether a putative endoplasmic reticulum retention code located in the carboxyl-terminus of the receptor's delta-subunit contributes to its retention. Site-directed mutagenesis will alter this code, and transport to the golgi apparatus will be monitored using dual- staining immunofluorescence. The third aim is to determine whether the association of calnexin with unassembled subunits provides protection from degradation. Calnexin usually recognizes glycoproteins by forming contracts with N-linked oligosaccharide chains, which can be removed by altering the signal Asn-X-Ser/Thr. All nicotinic acetylcholine receptor subunits possess a conserved glycosylation site at Asn-141. The Asn-141 glycosylation site will be altered and calnexin association, receptor accumulation, and transport to the endosomes will be assessed.